Hemoglobin I from Lucina pectinata is probably the only heme protein that carries out its function in the hydrogen-sulfide bound ferric state and in the dioxygen bound ferrous state. A proposed mechanism suggests that Hbl reacts with H2S to form a heme ferric sulfide low spin complex to transport the H2S to a bacteria symbion. Hydrogen sulfide is believed to be released to the bacteria upon formation of the ferrous protein by electron transfer from a still unknown reductant. Furthermore, the presence of H-bonding between the heme-Fe/+3 SH2 moiety and GInE7 is postulated to stabilize the HbISH2 center. Likewise, polar interactions between the PheB10 and PheE11 residues and the HbICO, HbIO2, and HBICN centers, respectively. This hypothesis is that Hbl may possesses two distinct ligand stabilization mechanism: one for the Hbl-SH2 center and the other for the HbICO, HBIO2, and HbICN moieties. The work proposed here is intended to unravel the interactions between the nearby amino acids and the HbISH2, HbIO2, HbICO, and HbICN moieties. A site-directed mutagenesis approach will be used. Choices of Hbl site-directed variants in the E7, B10, and E11 positions, and isotope labeling of ligands coupled to resonance and FTIR vibrational spectroscopies will clarify the Hbl-ligand dual stabilization mechanism and structure. We plan to address the factors that controls the binding, transport, and delivery of H2S by Hbl. Right now, there are intense arguments and discussions going on about whether the ligand affinity differences that are observed in heme proteins arise from electrostatic effects, from steric constraints, or from hydrogen-bonding differences. In the broad sense, Hbl offers a real advantage to address these issues as here the ferric, as well as the ferrous, protein is significant, unique and reversible when it carries its biological function. A long term goal of this project is to clarify the symbiotic interaction and communication between the aerobic Lucina pectinata and the unknown anaerobic bacteria.